Analytical chemists for many years did not use Raman spectroscopy because it failed to provide the sensitivity required for the detection of trace quantities of analytes. This was mainly because of very high background levels of fluorescence arising from either the sample or substrate which swamp the weak Raman signals. In 1974, Fleischman discovered, whilst using Raman spectroscopy to study the electrochemical reactions of pyridine on a silver electrode, that there was a tremendous enhancement of the pyridine Raman signals with the silver quenching a large amount of the background fluorescence. This surface enhancement could only be achieved if the silver surface was rough and not smooth.
Subsequently, it has been found that equal or even higher surface enhancement effects can be achieved with silver colloids. A colloid is a suspension of the metal particles in solution. In order to achieve the optimum effect, controlled aggregation of the silver colloid particles is required, typically using inorganic (e.g. chloride or nitrate) or organic (e.g. poly-L-lysine or spremine) compounds as aggregation reagents. For the majority of analytes, the colloidal silver particles should be about 20-50 nm, prior to aggregation, and have a narrow particle size distribution.
With the tremendous increase in sensitivity that can be achieved using this surface enhancement effects the analytical techniques of surface enhanced Raman scattering (SERS) spectroscopy and surface enhanced resonance Raman scattering (SERRS) spectroscopy have since been developed. The growth in the use of these techniques has been exponential but the major problem is producing stable colloids with good light scattering properties and capable of quenching background fluorescence. For a stable colloid the silver particles should remain suspended indefinitely but, on many occasions, aggregation occurs and the silver falls out of solution.
Silver colloids can be prepared by chemical reduction with either sodium borohydride or sodium citrate. Citrate reduced colloids are more stable and many analysts have prepared these using a method published by P. C. Lee and O. Meisel (J. Phys. Chem., 1982, 86, 3391-3395). Batch to batch reproducibility is difficult to achieve by this method and the stability, i.e. shelf life, is variable. Preparation requires the use of ultra clean glassware and accurately controlled temperatures, stirring speed, etc.
A published modification of the original method (C. H. Munro, W. E. Smith and P. C. White, Analyst 1993, Vol. 118, 733-735) has led to some improvements in the properties of the silver colloid but long term stability can still be a problem. For a commercial product, however greater stability is still required and studies have shown that even the material used in the storage container can affect the stability of some batches of silver colloids.
Silver colloids prepared according to these known prior methods produce silver particles with a negative citrate layer on their surface, and for maximum surface enhancement effects analytes must be in close proximity to the aggregated silver surface. For cationic analytes this can be achieved as they are attracted to the negatively charged silver surface. With anionic analytes only a very weak SER(R)S effect is achieved due to repulsion from the silver surface. The authors of the modified colloid preparation method resolved this problem by using poly-L-lysine as the aggregation agent, but adding ascorbic acid to control pH and protonate the aggregating agent, which then acted as a bridge between the colloid surface and the analyte.
In the search to achieve stable silver colloids with good light scattering properties, other methods of preparation have been studied by the Applicant. As a result of these studies a novel method, based upon knowledge of the chemical properties of inorganic materials, has resulted in producing stable so silver colloid solutions which have the desired analytical properties.